Soluble DNA is known to exist in the blood of healthy individuals at concentrations of about 5 to 10 ng/ml. It is believed that soluble DNA is present in increased levels in the blood of individuals having autoimmune diseases, particularly systemic lupus erythematosus (SLE) and other diseases including viral hepatitis, cancer and pulmonary embolism. It is not known whether circulating soluble DNA represents a specific type of DNA which is particularly prone to appear in the blood. However, studies indicate that the DNA behaves as double-stranded DNA or as a mixture of double-stranded and single-stranded DNA, and that it is likely to be composed of native DNA with single-stranded regions. Dennin, R. H., Klin. Wochenschr. 57:451-456, (1979). Steinman, C. R., J. Clin. Invest., 73:832-841, (1984). Fournie, G. J. et al., Analytical Biochem. 158:250-256, (1986). There is also evidence that in patients with SLE, the circulating DNA is enriched for human repetitive sequence (Alu) containing fragments when compared to normal human genomic DNA.
In patients with cancer, the levels of circulating soluble DNA in blood are significantly increased. Types of cancers which appear to have a high incidence of elevated DNA levels include pancreatic carcinoma, breast carcinoma, colorectal carcinoma and pulmonary carcinoma. In these forms of cancer, the levels of circulating soluble DNA in blood are usually over 50 ng/ml, and generally the mean values are more than 150 ng/ml. Leon et al., Can. Res. 37:646-650, 1977; Shapiro et al., Cancer 51:2116-2120, 1983.
Mutated oncogenes have been described in experimental and human tumors. In some instances certain mutated oncogenes are associated with particular types of tumors. Examples of these are adenocarcinomas of the pancreas, colon and lung which have approximately a 75%, 50%, and 35% incidence respectively, of Kirsten ras (K-ras) genes with mutations in positions 1 or 2 of codons 12. The most frequent mutations are changes from glycine to valine (GGT to GTT), glycine to cysteine (GGT to TGT), and glycine to aspartic acid (GGT to GAT). Other, but less common mutations of codon 12 include mutations to AGT and CGT. K-ras genes in somatic cells of such patients are not mutated.
The ability to detect sequences of mutated oncogenes or other genes in small samples of biological fluid, such as blood plasma, would provide a useful diagnostic tool. The presence of mutated K-ras gene sequences in the plasma would be indicative of the presence in the patient of a tumor which contains mutated oncogenes. Presumably this would be a specific tumor marker since there is no other known source of mutated K-ras genes. Therefore, this evaluation may be useful in suggesting and/or confirming a diagnosis. The amount of mutated K-ras sequences in the plasma may relate to the size of the tumor, the growth rate of the tumor and/or the regression of the tumor. Therefore, serial quantitation of mutated K-ras sequences may be useful in determining changes in tumor mass. Since most human cancers have mutated oncogenes, evaluation of plasma DNA for mutated sequences may have very wide applicability and usefulness.